PROJECT SUMMARY Infectious diseases of the oral cavity, such as dental caries and periodontal diseases, are associated with compositional changes in microbial communities, with certain microorganisms being more abundant in disease than in health, and vice versa. In the case of dental caries, the biofilm communities become enriched with acidogenic and acid tolerant species such as Streptococcus mutans, whereas health-associated biofilms are predominantly composed of commensal species, several of which are beneficial and can moderate biofilm acidification and interfere with the establishment and metabolic activities of caries pathogens. Many of the most abundant commensals persist by protecting themselves from low pH by breaking down substrates that yield alkaline products. Accumulating evidence now highlights the important role of alkali generation in oral biofilms, particularly through the production of ammonia from arginine via the arginine deiminase system. Recently, a novel oral Streptococcus strain, designated A12, was found to be highly arginolytic, but also potently antagonistic to S. mutans, thus having the ability to synergistically moderate biofilm pH and interfere with a ubiquitous caries pathogen. The focus of this research is to examine the molecular mechanisms by which the highly arginolytic, low-passage, clinical isolate, Streptococcus A12, competes against S. mutans; a primary goal being to dissect the genomic and physiologic basis for the phenotypic behaviors of this overtly beneficial organism using in vitro and in vivo models. Studying the beneficial properties of clinical isolates, such as A12, will help to establish a foundation for the design of new therapies and biomarkers to detect, diagnose and prevent dental caries. To accomplish this, the following aims are proposed: Aim 1) Examine the transcriptional organization and regulation of three genetic loci that contribute to the ability of Streptococcus A12 to compete with S. mutans, Aim 2) Evaluate the molecular mechanisms governing the interactions of A12 and S. mutans in vitro using dual-species biofilm models, and Aim 3) Evaluate the ability of Streptococcus A12 to inhibit colonization and/or persistence of S. mutans in an established mouse model. Aim 1 will examine the transcriptional organization and regulation of three genetic determinants that contribute to the ability of A12 to interfere with S. mutans. In Aim 2, the interaction of A12 and S. mutans will be evaluated in in vitro dual-species biofilm models and using RNA-seq to achieve a more comprehensive understanding of the interactions of A12 with S. mutans and the mechanisms by which the gene products of interest influence these interactions. In Aim 3, an established mouse model of dental caries will be employed to evaluate the capacity of A12 to inhibit the colonization and persistence of S. mutans. Collectively, this research will provide a comprehensive analysis of novel strategies employed by A12 to inhibit the dysbiosis that promotes oral diseases, thereby contributing important insights into probiotic effects of beneficial bacteria. The information gained will be of value in identifying other organisms that may promote oral health and guiding the development of effective probiotics.